Assessment of disulfide and hinge modifications in monoclonal antibodies

Abstract

During the last years there was a substantial increase in the use of antibodies and related proteins as therapeutics. The emphasis of the pharmaceutical industry is on IgG1, IgG2, and IgG4 antibodies, which are therefore in the focus of this article. In order to ensure appropriate quality control of such biopharmaceuticals, deep understanding of their chemical degradation pathways and the resulting impact on potency, pharmacokinetics, and safety is required. Criticality of modifications may be specific for individual antibodies and has to be assessed for each molecule. However, some modifications of conserved structure elements occur in all or at least most IgGs. In these cases, criticality assessment may be applicable to related molecules or molecule formats. The relatively low dissociation energy of disulfide bonds and the high flexibility of the hinge region frequently lead to modifications and cleavages. Therefore, the hinge region and disulfide bonds require specific consideration during quality assessment of mAbs. In this review, available literature knowledge on underlying chemical reaction pathways of modifications, analytical methods for quantification and criticality are discussed. The hinge region is prone to cleavage and is involved in pathways that lead to thioether bond formation, cysteine racemization, and iso-Asp (Asp, aspartic acid) formation. Disulfide or sulfhydryl groups were found to be prone to reductive cleavage, trisulfide formation, cysteinylation, glutathionylation, disulfide bridging to further light chains, and disulfide scrambling. With regard to potency, disulfide cleavage, hinge cleavage, disulfide bridging to further light chains, and cysteinylation were found to influence antigen binding and fragment crystallizable (Fc) effector functionalities. Renal clearance of small fragments may be faster, whereas clearance of larger fragments appears to depend on their neonatal Fc receptor (FcRn) functionality, which in turn may be impeded by disulfide bond cleavage. Certain modifications such as disulfide induced aggregation and heterodimers from different antibodies are generally regarded critical with respect to safety. However, the detection of some modifications in endogenous antibodies isolated from human blood and the possibility of in vivo repair mechanisms may reduce some safety concerns.

Keywords: Antibody; Critical quality attribute; Disulfide; Fragmentation; Modification.

Figure 1

CE‐SDS NGS separation of a non‐reduced IgG1. Attribution to underlying structures shows its high resolution for hinge cleavage and partially reduced forms.

Figure 2

Main fragmentation and modification processes in the upper hinge region. Mechanism 1 (A and B): Nucleophilic attack of serine OH on lysine carboxyl at pH 3–8 with subsequent hydrolysis (according to Vlasak et al. 11). Mechanism 2 (A and B): C‐terminal Asp cleavage is induced by protonation of the carbonyl of the peptide bond between Asp and Lys. Ring closure between the carbonyl carbon and the oxygen of the Asp carboxylgroup is succeeded by hydrolysis of the peptide bond (according to Vlasak et al. 11). This reaction is preferred under acidic conditions (below pH 5). Mechanism 3a (A and C): Abstraction of a proton from the nitrogen of the peptide bond between the upper hinge Asp and Lys and ring closure with the carbonyl of the Asp‐Cys peptide bond leads to an imidazoline derivative whose racemization leads to d‐Cys. Further β‐elimination of the disulfide bond and subsequent Michael addition leads to a thioether linkage. Mechanism 3b: Abstraction of a proton from the nitrogen of the peptide bond between the upper hinge Asp and Lys and ring closure with the carbonyl of the Asp side chain leads to a succinimide intermediate and subsequent hydrolysis to iso‐aspartate (according to Amano et al. 21). Mechanism 4 (A and B): Base attack leads to β‐elimination of the disulfide bond and formation of dehydroalanine. Subsequent hydrolysis leads to a noncovalent Fab amide and an Fab/c (pyruvoyl at the N‐terminus of the cleaved HC), whereas Michael‐like addition leads to Thioether formation (according to Vlasak et al. and Cohen et al. 11, 23). This reaction is preferred under basic conditions. Mechanism 5 (A): Radical attack of the C‐terminal upper hinge disulfide bond leads to sulfenic acid (Cys‐SOH) on one Cys and a thiyl radical (Cys‐S●) on the other 18. The thiyl radical transfers its electron to the upper hinge and induces backbone cleavage somewhere within the upper hinge. This forms Fab and Fab/c fragments with ragged termini 18, 23. The upper hinge histidine is assumed to be an important radical center during this process 19.

Figure 2

Main fragmentation and modification processes in the upper hinge region. Mechanism 1 (A and B): Nucleophilic attack of serine OH on lysine carboxyl at pH 3–8 with subsequent hydrolysis (according to Vlasak et al. 11). Mechanism 2 (A and B): C‐terminal Asp cleavage is induced by protonation of the carbonyl of the peptide bond between Asp and Lys. Ring closure between the carbonyl carbon and the oxygen of the Asp carboxylgroup is succeeded by hydrolysis of the peptide bond (according to Vlasak et al. 11). This reaction is preferred under acidic conditions (below pH 5). Mechanism 3a (A and C): Abstraction of a proton from the nitrogen of the peptide bond between the upper hinge Asp and Lys and ring closure with the carbonyl of the Asp‐Cys peptide bond leads to an imidazoline derivative whose racemization leads to d‐Cys. Further β‐elimination of the disulfide bond and subsequent Michael addition leads to a thioether linkage. Mechanism 3b: Abstraction of a proton from the nitrogen of the peptide bond between the upper hinge Asp and Lys and ring closure with the carbonyl of the Asp side chain leads to a succinimide intermediate and subsequent hydrolysis to iso‐aspartate (according to Amano et al. 21). Mechanism 4 (A and B): Base attack leads to β‐elimination of the disulfide bond and formation of dehydroalanine. Subsequent hydrolysis leads to a noncovalent Fab amide and an Fab/c (pyruvoyl at the N‐terminus of the cleaved HC), whereas Michael‐like addition leads to Thioether formation (according to Vlasak et al. and Cohen et al. 11, 23). This reaction is preferred under basic conditions. Mechanism 5 (A): Radical attack of the C‐terminal upper hinge disulfide bond leads to sulfenic acid (Cys‐SOH) on one Cys and a thiyl radical (Cys‐S●) on the other 18. The thiyl radical transfers its electron to the upper hinge and induces backbone cleavage somewhere within the upper hinge. This forms Fab and Fab/c fragments with ragged termini 18, 23. The upper hinge histidine is assumed to be an important radical center during this process 19.

Figure 2

Main fragmentation and modification processes in the upper hinge region. Mechanism 1 (A and B): Nucleophilic attack of serine OH on lysine carboxyl at pH 3–8 with subsequent hydrolysis (according to Vlasak et al. 11). Mechanism 2 (A and B): C‐terminal Asp cleavage is induced by protonation of the carbonyl of the peptide bond between Asp and Lys. Ring closure between the carbonyl carbon and the oxygen of the Asp carboxylgroup is succeeded by hydrolysis of the peptide bond (according to Vlasak et al. 11). This reaction is preferred under acidic conditions (below pH 5). Mechanism 3a (A and C): Abstraction of a proton from the nitrogen of the peptide bond between the upper hinge Asp and Lys and ring closure with the carbonyl of the Asp‐Cys peptide bond leads to an imidazoline derivative whose racemization leads to d‐Cys. Further β‐elimination of the disulfide bond and subsequent Michael addition leads to a thioether linkage. Mechanism 3b: Abstraction of a proton from the nitrogen of the peptide bond between the upper hinge Asp and Lys and ring closure with the carbonyl of the Asp side chain leads to a succinimide intermediate and subsequent hydrolysis to iso‐aspartate (according to Amano et al. 21). Mechanism 4 (A and B): Base attack leads to β‐elimination of the disulfide bond and formation of dehydroalanine. Subsequent hydrolysis leads to a noncovalent Fab amide and an Fab/c (pyruvoyl at the N‐terminus of the cleaved HC), whereas Michael‐like addition leads to Thioether formation (according to Vlasak et al. and Cohen et al. 11, 23). This reaction is preferred under basic conditions. Mechanism 5 (A): Radical attack of the C‐terminal upper hinge disulfide bond leads to sulfenic acid (Cys‐SOH) on one Cys and a thiyl radical (Cys‐S●) on the other 18. The thiyl radical transfers its electron to the upper hinge and induces backbone cleavage somewhere within the upper hinge. This forms Fab and Fab/c fragments with ragged termini 18, 23. The upper hinge histidine is assumed to be an important radical center during this process 19.

Figure 3

Formation of functional covalent dimers between identical as well as different IgG2 molecules.

Figure 4

From left to right: IgG2‐A, IgG2‐A/B, and IgG2‐B isoforms.

Figure 5

Exchange of Fab arms between endogenous and therapeutic IgG4 molecules.